This application is a divisional of U.S. patent application Ser. No. 09/545,737, filed Apr. 10, 2000, now U.S. Pat. No. 6,387,890, which is itself a continuation-in-part of International Patent Application PCT/US98/21430, published in the English language on Oct. 9, 1998, and is entitled to priority pursuant to 35 U.S.C. xc2xa7 119(e) to U.S. provisional patent application No. 60/061,607, filed Oct. 10, 1997.
The invention relates generally to enzyme inhibitors, more particularly to inhibitors of the enzyme designated arginase.
Each individual excretes roughly ten kilograms of urea per year, as a result of the hydrolysis of arginine in the final cytosolic step of the urea cycle (Krebs et al., 1932, Hoppe-Seyler""s Z. Physiol. Chem. 210:33-66). The activity of the liver enzyme, arginase, permits disposal of nitrogenous wastes which result from protein catabolism (Herzfeld et al., 1976, Biochem. J. 153:469-478). In tissues which lack a complete complement of the enzymes which catalyze the reactions of the urea cycle, arginase regulates cellular concentrations of arginine and ornithine, which are used for biosynthetic reactions (Yip et al., 1972, Biochem. J. 127:893-899). Arginine is used, by way of example, in the synthesis of nitric oxide. In macrophages, arginase activity is reciprocally coordinated with the activity of the enzyme, nitric oxide synthase. Reciprocal coordination of the activities of arginase and nitric oxide (NO) synthase modulates NO-dependent cytotoxicity (Corraliza et al., 1995, Biochem. Biophys. Res. Commun. 206:667-673; Daghigh et al., 1994, Biochem. Biophys. Res. Commun. 202:174-180; Chxc3xa9nais et al., 1993, Biochem. Biophys. Res. Commun. 196:1558-1565; Klatt et al., 1993, J. Biol. Chem. 268:14781-14787; Keller et al., 1991, Cell. Immunol. 134:249-256; Albina et al., 1995, J. Immunol. 155:4391-4396).
Synthesis and evaluation of non-reactive arginine analogs for use as enzyme inhibitors or receptor antagonists is a rapidly growing area of medicinal chemical research (Griffith et al., 1995, Annu. Rev. Physiol. 57:707-736; Gross et al., 1990, Biochem. Biophys. Res. Commun. 170:96-103; Hibbs et al., 1987, J. Immunol. 138:550-565; Lambert et al., 1991, Life Sci. 48:69-75; Olken et al., 1992, J. Med. Chem. 35:1137-1144; Feldman et al., 1993, J. Med. Chem. 36:491-496; Narayanan et al., 1994, FASEB J. 8:A360; Narayanan et al., 1994, J. Med. Chem. 37:885-887; Moore et al., 1994, J. Med. Chem. 37:3886-3888; Moynihan et al., 1994, J. Chem. Soc. Perkin Trans.769-771; Robertson et al., 1995, J. Bioorganic Chem. 23:144-151).
To date, the X-ray crystal structure of one of the enzymes of mammalian arginine catabolism, namely rat liver arginase, is available (Kanyo et al., 1996, Nature 383:554-557). Rat liver arginase is a trimeric metalloenzyme which contains a bi-nuclear manganese cluster in the active site of each subunit. This bi-nuclear cluster is required for maximal catalytic activity (Reczkowski et al., 1992, J. Am. Chem. Soc. 114:10992-10994).
As noted herein, arginase catalyzes divalent cation-dependent hydrolysis of L-arginine to form L-omithine and urea. The enzyme is currently known to serve three important functions: production of urea, production of ornithine, and regulation of substrate arginine levels for nitric oxide synthase (Jenkinson et al., 1996, Comp. Biochem. Physiol. 114B:107-132; Kanyo et al., 1996, Nature 383:554-557; Christianson, 1997, Prog. Biophys. Molec. Biol. 67:217-252). Urea production provides a mechanism to excrete nitrogen in the form of a highly soluble, non-toxic compound, thus avoiding the potentially dangerous consequences of high ammonia levels. L-ornithine is a precursor for the biosynthesis of polyamines, spermine, and spermidine, which have important roles in cell proliferation and differentiation. Finally, arginase modulates production of nitric oxide by regulating the levels of arginine present within tissues.
Since both NO synthase and arginase compete for the same substrate, the possibility of reciprocal regulation of both arginine metabolic pathways has recently been explored (Modelell et al., 1995, Eur. J. Immunol. 25:1101-1104; Wang et al., 1995, Biochem. Biophys. Res. Commun. 210:1009-1016). Furthermore, N107-hydroxy-L-arginine (L-HO-Arg), an intermediate in the NO synthase reaction (Pufahl et al., 1992, Biochemistry 31:6822-6828; Klau et al, 1993, J. Biol. Chem. 268:14781-14787; Furchgom, 1995, Annu. Rev. Pharmacol. Toxicol., 35:1-27; Yamaguchi et al., 1992, Eur. J. Biochem., 204:547-552; Pufahl et al., 1995, Biochemistry 34:1930-1941), is an endogenous arginase inhibitor (Chenais et al., 1993, Biochem. Biophys. Res. Commun., 196:1558-1565; Buga et al., 1996, Am. J. Physiol. Heart Circ. Physiol. 271:H1988-H1998; Daghigh et al., 1994, Biochem. Biophys. Res. Commun. 202:174-180; Boucher et al., 1994, Biochem. Biophys. Res. Commun. 203:1614-1621). The phenomenon of reciprocal regulation between arginase and NO synthase has only recently been examined (Chakder and Rattan, 1997, J. Phannacol. Exp. Ther. 282:378-384; Langle et al., 1997, Transplantation 63:1225-1233; Langle et al., 1995, Transplantation 59:1542-1549). In the internal anal sphincter (IAS), it was shown that exogenous administration of arginase attenuates NO synthase-mediated non-adrenergic and non-cholinergic (NANC) nerve-mediated relaxation (Chakder and Rattan, 1997, J. Pharmacol. Exp. Ther. 282:378-384).
An excess of arginase has recently been associated with a number of pathological conditions that include gastric cancer (Wu et al., 1992, Life Sci. 51:1355-1361; Leu and Wang, 1992, Cancer 70:733-736; Straus et al., 1992, Clin. Chim. Acta 210:5-12; Ikemoto et al, 1993, Clin. Chem. 39:794-799; Wu et al., 1994, Dig. Dis. Sci. 39:1107-1112), certain forms of liver injury (Ikemoto et al., 1993, Clin. Chem. 39:794-799), and pulmonary hypertension following the orthotopic liver transplantation (Langle et al., 1997, Transplantation 63:1225-1233; Langle et al., 1995, Transplantation 59:1542-1549). Furthermore, high levels of arginase can cause impairment in NANC-mediated relaxation of the IAS (Chakder and Rattan, 1997, J. Pharmacol. Esp. Ther. 282:378-384). Previous studies have demonstrated that arginase pre-treatment causes significant suppression of the NANC nerve-mediated relaxation of the IAS (Chakder and Rattan 1997, J. Pharmacol. Exp. Ther. 282:378-384) that is mediated primarily via the L-arginine-NO synthase pathway (Rattan and Chakder, 1992, Am. J. Physiol. Gastrointest. Liver Physiol. 262:G107-G112; Rattan and Chakder, 1992, Gastroenterology 103:43-50). Impairment in NANC relaxation by excess arginase may be related to L-arginine depletion (Wang et al., 1995, Eur. J. Immunol. 25:1101-1104). Furthermore, suppressed relaxation could be restored by the arginase inhibitor L-HO-Arg. It is possible, therefore, that patients with certain conditions associated with an increase in arginase activity may stand to benefit from treatment with arginase inhibitors. However, an arginase inhibitor such as L-OH-Arg can not be selective since it also serves as a NO synthase substrate (Pufahl et al., 1992, Biochemistry 31:6822-6828; Furchgott, 1995, Annu. Rev. Pharmacol. Toxicol. 25:1-27; Pufahl et al, 1995, Biochemistry 34:1930-1941; Chemais et al., 1993, Biochem. Biophys. Res. Commun. 196:1558-1565; Boucher et al., 1994, Biochem. Biophys. Res. Commun. 203:1614-1621; Griffith and Stuehr, 1995, Annu. Rev. Physiol. 57:707-736). Because of this, the exact role of arginase in pathophysiology and the potential therapeutic actions of arginase inhibitors remains undetermined.
Erectile dysfunction afflicts one-half of the male population over the age of forty. This malady often results from defects in the complex cascade of enzyme-catalyzed reactions governing blood flow into and out of the corpus cavernosum, a chamber of muscular, spongy tissue that becomes engorged with blood in the erect penis. Defects that compromise cavernosal blood flow often occur as secondary complications related to other health conditions, such as heart disease, hypertension, diabetes, use of certain medications, and the like.
A need remains for inhibitors of arginase activity, which are useful for treating diseases or disorders characterized either by abnormally high arginase activity in a tissue of a mammal or by abnormally low nitric oxide synthase activity in a tissue of the mammal.
The invention include a composition comprising an arginase inhibitor having the structure
HOOCxe2x80x94CH(NH2)xe2x80x94X1xe2x80x94X2xe2x80x94X3xe2x80x94X4xe2x80x94B(OH)2 
wherein each of X1, X2, X3, and X4 is selected from the group consisting of xe2x80x94(CH2)xe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94(NH)xe2x80x94, and xe2x80x94(N-alkyl)-, except X2 is not xe2x80x94Sxe2x80x94when each of X1, X3, and X4 is xe2x80x94(CH2)xe2x80x94. In a preferred embodiment, the inhibitor is 2(S)-amino-6-boronohexanoic acid (ABHA), which has the structure
HOOCxe2x80x94CH(NH2)xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94B(OH)2. 
The composition can further comprise a pharmaceutically acceptable carrier.
Also included in the invention is a pharmaceutical composition comprising a pharmaceutical acceptable carrier and an arginase inhibitor having the structure
HOOCxe2x80x94CH(NH2)xe2x80x94X1xe2x80x94X2xe2x80x94X3xe2x80x94X4xe2x80x94B(OH)2 
wherein each of X1, X2, X3, and X4 is selected from the group consisting of xe2x80x94(CH2)xe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94(NH)xe2x80x94, and xe2x80x94(N-alkyl)-. For example, the structure can be one of
HOOCxe2x80x94CH(NH2)xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94B(OH)2 and 
HOOCxe2x80x94CH(NH2)xe2x80x94CH2xe2x80x94Sxe2x80x94CH2xe2x80x94CH2xe2x80x94B(OH)2. 
The invention further includes a method of inhibiting arginase. This method comprises contacting the arginase with a compound having the structure
HOOCxe2x80x94CH(NH2)xe2x80x94X1xe2x80x94X2xe2x80x94X3xe2x80x94X4xe2x80x94B(OH)2 
wherein each of X1, X2, X3, and X4 is selected from the group consisting of xe2x80x94(CH2)xe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94(NH)xe2x80x94, and xe2x80x94(N-alkyl)-. The arginase can, for example, be a yeast arginase or a mammalian arginase. When the arginase is mammalian arginase, the arginase is human arginase such as a human type II arginase (e.g. human penile arginase).
Also included in the invention is a method of inhibiting arginase in a mammal. This method comprises administering to the mammal (e.g. a human) a composition comprising a pharmaceutically acceptable carrier and an arginase inhibitor having the structure
HOOCxe2x80x94CH(NH2)xe2x80x94X1xe2x80x94X2xe2x80x94X3xe2x80x94X4xe2x80x94B(OH)2 
wherein each of X1, X2, X3, and X4 is selected from the group consisting of xe2x80x94(CH2)xe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94(NH)xe2x80x94, and xe2x80x94(N-alkyl)-. The structure can, for example, be either of
HOOCxe2x80x94CH(NH2)xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94B(OH)2 and 
HOOCxe2x80x94CH(NH2)xe2x80x94CH2xe2x80x94Sxe2x80x94CH2xe2x80x94CH2xe2x80x94B(OH)2. 
When the mammal is a human, the human can be one who comprises either a tissue which exhibits an abnormally high level of arginase activity or a tissue which exhibits an abnormally low level of nitric oxide synthase activity.
Also included in the invention is a method of treating an arginase-related disorder in a human. This method comprises administering to the human a composition comprising a pharmaceutically acceptable carrier and an arginase inhibitor having the structure
HOOCxe2x80x94CH(NH2)xe2x80x94X1xe2x80x94X2xe2x80x94X3xe2x80x94X4xe2x80x94B(OH)2 
wherein each of X1, X2, X3, and X4 is selected from the group consisting of xe2x80x94(CH2)xe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94(NH)xe2x80x94, and xe2x80x94(N-alkyl)-. The disorder can, for example, be one selected from the group consisting of a disorder associated with an abnormally low level of nitric oxide synthase activity in a tissue of the human and a disorder associated with an abnormally high level of arginase activity in a tissue of the human. Examples of such disorders include heart disease, systemic hypertension, pulmonary hypertension, erectile dysfunction, autoimmune encephalomyelitis, chronic renal failure, gastrointestinal motility disorders, gastric cancers, reduced (or insufficient) hepatic blood flow, and cerebral vasospasm.
The invention further includes a method of relaxing smooth muscle in a mammal. This method comprises administering to the mammal a composition comprising a pharmaceutically acceptable carrier and an arginase inhibitor having the structure
HOOCxe2x80x94CH(NH2)xe2x80x94X1xe2x80x94X2xe2x80x94X3xe2x80x94X4xe2x80x94B(OH)2 
wherein each of X1, X2, X3, and X4 is selected from the group consisting of xe2x80x94(CH2)xe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94(NH)xe2x80x94, and xe2x80x94(N-alkyl)-. The smooth muscle which is relaxed according to this method can be one of a gastrointestinal smooth muscle, anal sphincter smooth muscle, esophageal sphincter muscle, corpus cavernosum, sphincter of Oddi, arterial smooth muscle, heart smooth muscle, pulmonary smooth muscle, kidney smooth muscle, uterine smooth muscle, vaginal smooth muscle, cervical smooth muscle, placental smooth muscle, and ocular smooth muscle.
In addition, the invention includes a method of making a compound having the structure
HOOCxe2x80x94CH(NH2)xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94B(OH)2. 
This method comprises contacting a molecule of the tert-butyl ester of 2(S)-N-(tert-butyloxycarbonyl)-6-[(1S,2S,3R,5S)-(+)-pinanedioxaboranyl]-hexanoic acid in an organic solvent (e.g. CH2Cl2) with BCl3.
Also included in the invention is a method of making a compound having the structure
HOOCxe2x80x94CH(NH2)xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94B(OH)2. 
This method comprises the steps of
(a) mixing a solution of the tert-butyl ester of 2(S)-N-(tert-butyloxycarbonyl)-glutamic acid in tetrahydrofuran with triethylamine and ethyl chloroformate to produce a first mixture, removing the resulting triethylammonium hydrochloride salt by filtration, and treating the remaining mixture with an aqueous solution of sodium borohydride to provide a first compound, wherein the first compound is the tert-butyl ester of 2(S)xe2x80x94N-(tert-butyloxycarbonyl)-5-hydroxypentanoic acid;
(b) subjecting the first compound to Swem oxidation to produce a second compound;
(c) subjecting the second compound to a Wittig reaction in the presence of triphenylphosphonium methylide to produce a third compound;
(d) mixing a solution of BH3 with the third compound in the presence of tetrahydrofuran to produce a second mixture;
(e) adding (1S,2S,3R,5S)-(+)-pinanediol to the second mixture to produce a fourth compound, wherein the fourth compound is the tert-butyl ester of 2(S)-N-(tert-butyloxycarbonyl)-6-[(1S,2S,3R,5S)-(+)-pinanedioxaboranyl]-hexanoic acid; and
(f) mixing the fourth compound with BCl3 in the presence of CH2Cl2 to produce the compound.
Also included in the invention is a method of identifying an arginase inhibitor antagonist, the method comprising the steps of
(a) inducing relaxation of a muscle in vitro;
(b) reversing the relaxation by contacting the muscle with arginase;
(c) adding an arginase inhibitor to the muscle so reversed to renew relaxation of the muscle in the presence or absence of a test compound; and
(d) measuring the level of renewed relaxation of the muscle, wherein a lower level of renewed relaxation of the muscle in the presence of the test compound, compared with the level of renewed relaxation of the muscle in the absence of the test compound, is an indication that the test compound is an arginase inhibitor antagonist.
In another aspect, the invention relates to a method of alleviating erectile dysfunction in a human. In this method, a pharmaceutical composition is administered to the human, the composition comprising an arginase inhibitor having the structure
HOOCxe2x80x94CH(NH2)xe2x80x94X1xe2x80x94X2xe2x80x94X3xe2x80x94X4xe2x80x94B(OH)2 
wherein each of X1, X2, X3, and X4 is selected from the group consisting of xe2x80x94(CH2)xe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94(NH)xe2x80x94, and xe2x80x94(N-alkyl)-. Preferably, the arginase inhibitor is ABHA.